This invention is in the field of measurement of microbiological activity in highly scattering systems. Specifically, this patent application is in the field of fluorescent measurement of microbiological activity in opaque mediums such as slurries and colloids and certain Metal Working Fluids.
Microbial contamination in opaque mediums such as slurries and colloids and certain Metal Working Fluids is a significant problem in many industries. In papermaking, additives such as kaolin slurry, precipitated calcium carbonate suspensions or starch solutions can harbor large microbial populations, which serve as inocullum for the papermnachine. Mining companies are required to supply industries such as paper and ceramics with treated and preserved additives and also need to monitor microbial contamination. Certain Metal Working Fluids are also susceptible to microbiological contamination.
The conventional method of controlling microbial growth is through the use of biocides. Biocides are chemicals that inhibit microbial growth by destroying the cell wall or cellular constituents of microorganisms. Physical conditions such as temperature, radiation, or interactions with treatment chemicals contained within a system can have a negative impact on the effectiveness of the biocide. To compensate for the reduced effect, biocides can either be added continuously or intermittently on an xe2x80x9cas-requiredxe2x80x9d basis. The minimal possible use of biocides is encouraged since biocides are both expensive and toxic. Thus, to prevent waste, constant monitoring and testing of the slurry or colloid or Metal Working Fluid is required to determine the proper quantity of biocide for controlling microbial growth.
Most slurries and colloids and certain Metal Working Fluids are opaque, which means they are not transparent to the passage of light. Another way to describe opaque media is that they are highly light scattering media. For the purposes of this patent application the term, xe2x80x9copaquexe2x80x9d is used to refer to any medium which when placed in a 1 cm cuvette in the path of a non-absorbing visible light beam, acts to reduce the intensity of the light by 20% or more due to scattering.
When media are opaque, it is not possible to know what is inside an opaque media simply by looking at it. This means that it is impossible to tell if there is microbiological contamination of an opaque slurry or an opaque colloid or an opaque Metal Working Fluid by looking at it. Therefore, conventional, known optical methods of detection of microbiological contamination (such as optical density measurements and ATP measurements) cannot give results for opaque media. This is because light cannot pass through the sample, as light loss becomes inversely proportional to the extent of light scattering. Therefore, other methods of detecting microbial contamination in an opaque media must be used.
At present samples of opaque slurries or opaque colloids or opaque Metal Working Fluids are typically monitored for microbiological contamination using standard xe2x80x9cplate-countxe2x80x9d methods. Standard xe2x80x9cplate-countxe2x80x9d methods are typically referred to as xe2x80x9cplatingxe2x80x9d. Plating of samples requires trained personnel, equipment and a 48 hours incubation period during which microbes in the slurry can reproduce rampantly. The actual method of plate counting involves withdrawing a sample, diluting the sample, and applying the sample to the surface of a Nutrient agar medium. After incubation for 24 to 48 hours, the sample is checked for the presence of microorganisms and, where appropriate, the organisms are counted by manual or video means.
Some industrial situations require the use of High Pressure Liquid Chromatographs (HPLC) to determine if there is residual biocide left in the sample. HPLC can only measure biocide concentration and not microbial activity. HPLC also requires expensive equipment and trained personnel for routine measurements. Since HPLC only measures residual biocides, it cannot measure biocide resistant strains of microbiological organisms that are developing in the opaque media.
In addition to grab sampling, other on-site sampling techniques are available, such as Dip slide and Adenosine Triphosphate (ATP) tests. Unfortunately, such tests are not practical to use when measuring microbiological contamination in opaque medium because ATP tests require a transparent sample and therefore do not work in opaque medium and Dip slides require 24 to 48 hours for test results to develop. Thus, neither test is suitable for field evaluation of microbiological contamination.
Additional methods for monitoring the microbial populations in various mediums are described and claimed in the following references.
U.S. Pat. No. 5,206,151 describes and claims a method to measure the minimum inhibitory concentration of biocides by adding various amounts, types and combinations of biocides to aliquots of sample containing bacteria, adding an oxidation-reduction dye such as Resazurin or tetrazolium violet and Nutrients and monitoring the change in color.
U.S. Pat. No. 5,413,916 describes and claims a method for determination of toxicity of an environmental sample to bacteria by the addition of Resazurin and glutaraldehyde and bacteria to the sample and measuring absorbance (at 603 nm) as compared to a blank.
U.S. Pat. No. 5,336,600 describes and claims a method for detection of micro-organisms consisting of mixing a Resorufin (or Resorufin derivative not including Resazurin) and Nutrient medium and measuring a decrease in the fluorescence.
U.S. Pat. No. 5,523,214 describes and claims a method of identification of microbes using methylene blue and Resazurin stabilized with potassium ferricyanate or iron salts mixed with potassium ferrocyanate or sodium tungsate or tartrazine yellow or reactive red 4 or similar compounds. The patent claims substantial increase in sensitivity using this mixture as compared to using either dye alone.
Aliquots of sand filters were assessed using Resazurin reduction method, in an experiment described in an article entitled: xe2x80x9cResazurin reduction tests as an estimate of coliform and heterotrophic bacterial numbers in environmental samplesxe2x80x9d Can. Bull. Environ. Contam. Toxicol. 49, 354, 1992.
An article entitled, xe2x80x9cResazurin reduction as a function of respiratory burst in bovine neutrophils is an article in Am. J. Vet. Res. 58, 601, 1997, describes a technique of fluorometrically monitoring the end-point of Resazurin (Resorufin) as a measure of respiratory burst.
The article, xe2x80x9cAutomation of the Resazurin Reduction Test using Fluorometry of Microtitration Traysxe2x80x9d, by Ali-Vehmas, Louhi and Sandholm, J. Vet. Med., B 38, 358-372 (1991) describes the automation of the fluorescent Resazurin-to-Resorufin reduction test for monitoring bacterial numbers in broth cultures and milk. The reduction of Resazurin (blue color) to Resorufin (a pink color) and finally and reversibly to dihydroresorufin (colorless) is well known in the art of determining mircrobiological contamination in milk. This method involves taking contaminated samples of milk in microtitration plates, adding a Nutrient medium and measuring the fluorescence corresponding to the Resorufin peak at 5 min intervals. This continuous measurement of the same sample makes this an automated measurement. The Resorufin intensity peaks when the increase in intensity due to conversion from Resazurin is offset by the decrease due to formation of the non-fluorescent dihydroresorufin. In this work, the sample population is increased significantly by addition of Nutrient medium (which is a necessary part of the method).
U.S. Pat. No. 6,060,318, entitled, xe2x80x9cTracing of Process Additives in Industrial Ceramics Applicationsxe2x80x9d, claims a fluorometric method for monitoring concentration of chemicals in ceramic slurries and powders having an external surface. In this patent, a solid-state fluorometer, (in the surface fluorescence configuration) is used to monitor the concentration of fluorescence molecules in ceramic slurries. Applications within ceramic slurries include monitoring of treatment dosages; measurement of mixing times in batch mixing vessels; determination of batch contamination from ball mills and other mixing vessels; and, efficiency of transfer from ball mills to mixing tanks.
It would be desirable to have an alternative method developed to determine the level of microbiological contamination in opaque slurries and opaque colloidal materials and opaque Metal Working Fluids.
The first aspect of the instant claimed invention is a process for monitoring of microbiological populations in an opaque medium comprising:
a) obtaining an Aliquot of material from the opaque medium;
b) adding a Fluorogenic Dye to said Aliquot, wherein said Aliquot is now referred to as Aliquot-Dye;
c) allowing said Fluorogenic Dye to react with any microbiological organisms present;
d) providing means for measurement of the fluorescent signals of the Fluorogenic Dye and the Reacted Fluorogenic Dye in said Aliquot-Dye;
e) using said means for measurement to measure the fluorescent signals of the Fluorogenic Dye and the Reacted Fluorogenic Dye, while discarding any measured fluorescent signal values below a predetermined noise level;
f) calculating the RATIO of the fluorescent signal of the Reacted Fluorogenic Dye to the fluorescent signal of the Fluorogenic Dye; and
g) using said RATIO to monitor the extent of microbiological contamination in said opaque medium.
The second aspect of the instant claimed invention is the process of the first aspect of the instant claimed invention further comprising:
h) using said RATIO to determine the optimal amount of biocide to deliver to the opaque medium; and
i) delivering said optimal amount of biocide to the opaque medium.
The third aspect of the instant claimed invention is a process for monitoring of microbiological populations in an opaque medium comprising:
(A) separating at least two Aliquots of material, optionally three Aliquots of material, from the opaque medium;
(B) adding nothing to the first Aliquot, wherein said first Aliquot is now referred to as Aliquot-Blank, adding a Fluorogenic Dye to the second Aliquot, wherein said second Aliquot is now referred to as Aliquot-Dye, and if the optional third Aliquot is present, adding a Metabolic Inhibitor to the optional third Aliquot, followed by adding Fluorogenic Dye to the optional third Aliquot, wherein said third Aliquot is now referred to as optional Aliquot-Inhibitor-Dye;
(C) allowing said Fluorogenic Dye to react with any microbiological organisms present;
(D) providing means for measurement of the fluorescent signals in said Aliquot-Blank, in said Aliquot-Dye, and in said optional Aliquot-Inhibitor-Dye, with the fluorescent signals being measured at the wavelength of the Fluorogenic Dye and at the wavelength of the Reacted Fluorogenic Dye;
(E) using said means for measurement of said fluorescent signals to measure the fluorescent signals in Aliquot-Blank, Aliquot-Dye, and in optional Aliquot-Inhibitor-Dye, at the wavelength of the Fluorogenic Dye and at the wavelength of the Reacted Fluorogenic Dye, while discarding any measured fluorescent signal values below a predetermined noise level;
(F) calculating the Useful RATIO, wherein the Useful RATIO is selected from the group consisting of RATIO of Adjusted for Background Fluorescence Fluorescent Signal of the Reacted Fluorogenic Dye to the Adjusted for Background Fluorescence Fluorescent Signal of the Fluorogenic Dye and RATIO of the Adjusted for Interactions with chemicals and Background Fluorescence Fluorescent Signal of the Reacted Fluorogenic Dye to the Adjusted for Interactions with chemicals and Background Fluorescence Fluorescent Signal of the Fluorogenic Dye;
(G) using the Useful RATIO to monitor the extent of microbiological contamination in said opaque medium.
The fourth aspect of the instant claimed invention is the process of the third aspect of the instant claimed invention further comprising:
(H) using one or both of the Useful RATIOs from steps (F) and (G) to determine the optimal amount of biocide to deliver to said opaque medium; and
(I) delivering said optimal amount of biocide to the opaque medium.
The fifth aspect of the instant claimed invention is a process for monitoring of microbiological populations in an opaque medium comprising:
a) obtaining an Aliquot of material from the opaque medium;
b) adding a Fluorogenic Dye into said Aliquot, wherein said Aliquot is now referred to as Aliquot-Dye;
c) allowing said Fluorogenic Dye to react with any microbiological organisms present for a time period known as Time Zero;
d) providing means for measurement of the fluorescent signals of the Fluorogenic Dye and the Reacted Fluorogenic Dye in said Aliquot-Dye;
e) using said means for measurement of said fluorescent signals to measure the fluorescent signals of the Fluorogenic Dye and the Reacted Fluorogenic Dye at Time Zero, while discarding any measured fluorescent signal values below a predetermined noise level;
f) calculating the RATIO of the fluorescent signal of the Reacted Fluorogenic Dye to the fluorescent signal of the Fluorogenic Dye and designating that RATIO the RATIO at Time Zero;
g) waiting for a time period, designated Time Future,
h) measuring the fluorescent signals of the Fluorogenic Dye and the Reacted Fluorogenic Dye in Aliquot-Dye at Time Future;
i) calculating the RATIO of the fluorescent signal of the Reacted Fluorogenic Dye at Time Future to the fluorescent signal of the Fluorogenic Dye at Time Future, designating that RATIO the RATIO at Time Future;
j) comparing the RATIO at Time Future to the RATIO at Time Zero; and
k) using the comparison of the RATIO at Time Future to the RATIO at Time Zero to monitor the extent of microbiological contamination in said opaque medium.
The sixth aspect of the instant claimed invention is the process of the fifth aspect of the instant claimed invention further comprising:
l) using the comparison of the RATIO at Time Future to the RATIO at Time Zero to determine the optimal amount of biocide to deliver to said opaque medium; and
m) delivering said optimal amount of biocide to the opaque medium.
The seventh aspect of the instant claimed invention is a process for monitoring of microbiological populations in an opaque medium comprising:
(A) separating at least two Aliquots of material, optionally three Aliquots of material, from the opaque medium;
(B) adding nothing to the first Aliquot, wherein said first Aliquot is now referred to as Aliquot-Blank, adding a Fluorogenic Dye to the second Aliquot, wherein said second Aliquot is now referred to as Aliquot-Dye, and if the optional third Aliquot is present, adding a Metabolic Inhibitor followed by a Fluorogenic Dye to the optional third Aliquot, wherein the optional third Aliquot is now referred to as optional Aliquot-Inhibitor-Dye;
(C) allowing said Fluorogenic Dye to react with any microbiological organisms present for a time period known as Time Zero;
(D) providing means for measurement of the fluorescent signals in said Aliquot-Blank, in said Aliquot-Dye, and in said optional Aliquot-Inhibitor-Dye, with the fluorescent signals being measured at the wavelength of the Fluorogenic Dye and at the wavelength of the Reacted Fluorogenic Dye;
(E) using said means for measurement of said fluorescent signals to measure the fluorescent signals in Aliquot-Blank, Aliquot-Dye and in optional Aliquot-Inhibitor-Dye at Time Zero, at the wavelength of the Fluorogenic Dye and the wavelength of the Reacted Fluorogenic Dye, while discarding any measured fluorescent signal values below a predetermined noise level to yield fluorescent signals at Time Zero;
(F) calculating the Useful RATIO at Time Zero, wherein the Useful RATIO at Time Zero is selected from the group consisting of RATIO at Time Zero of the Adjusted for Background Fluorescence Fluorescent Signal of the Reacted Fluorogenic Dye to the Adjusted for Background Fluorescence Fluorescent Signal of the Fluorogenic Dye at Time Zero and optional RATIO of the Adjusted for Interactions with chemicals and Background Fluorescence Fluorescent Signal of the Reacted Fluorogenic Dye to the Adjusted for Interactions with chemicals and Background Fluorescence Fluorescent Signal of the Fluorogenic Dye,
(G) waiting for a time period, designated Time Future;
(H) using said means for measurement to measure the fluorescent signals at Time Future in Aliquot-Blank, Aliquot-Dye and in optional Aliquot-Inhibitor-Dye at the wavelength of the Fluorogenic Dye and the wavelength of the Reacted Fluorogenic Dye;
(I) calculating the Useful RATIO at Time Future, wherein the Useful RATIO at Time Future is selected from the group consisting of RATIO at Time Future of the Adjusted for Background Fluorescence Fluorescent Signal of the Reacted Fluorogenic Dye to the Adjusted for Background Fluorescence Fluorescent Signal of the Fluorogenic Dye at Time Future and optional RATIO at Time Future of the Adjusted for Interactions with chemicals and Background Fluorescence Fluorescent Signal of the Reacted Fluorogenic Dye to the Adjusted for Interactions with chemicals and Background Fluorescence Fluorescent Signal of the Fluorogenic Dye;
(J) comparing the Useful RATIO at Time Future to the RATIO at Time Zero; and
(K) using the comparison of the Useful RATIO at Time Future to the RATIO at Time Zero to monitor the extent of microbiological contamination in said opaque medium.
The eighth aspect of the instant claimed invention is the process of the seventh aspect of the instant claimed invention further comprising:
(L) using the comparison of the Useful RATIO at Time Future to the Useful RATIO at Time Zero to determine the optimal amount of biocide to deliver to said opaque medium; and
(M) delivering said optimal amount of biocide to the opaque medium.
The ninth aspect of the instant claimed invention is a process for monitoring both active and inactive microbiological populations in an opaque medium, optionally accounting for chemical interference with the test method, as well as optionally accounting for background fluorescence comprising:
(A) obtaining two Aliquots of material, optionally three or four Aliquots of material from the opaque medium;
(B) adding a Fluorogenic Dye directly into the first Aliquot, wherein the first Aliquot is now referred to as Aliquot-Dye, adding Nutrient and Fluorogenic Dye to the second Aliquot, wherein the second Aliquot is now referred to as Aliquot-Nutrient-Dye, if the optional third Aliquot is present, adding a Metabolic Inhibitor and Fluorogenic Dye to the optional third Aliquot, wherein the optional third Aliquot is now referred to as optional Aliquot-Inhibitor-Dye, and if the optional fourth Aliquot is present, adding nothing to the fourth Aliquot, wherein the fourth Aliquot is now referred to as optional Aliquot-Blank;
(C) allowing said Fluorogenic Dye to react with any microbiological organisms present for a time period known as Time Zero;
(D) providing means for measurement of the fluorescent signals in said Aliquot-Dye, said Aliquot-Nutrient-Dye, said optional Aliquot-Inhibitor-Dye and in said optional Aliquot-Blank, with the fluorescent signals in each Aliquot being measured at the wavelength of the Fluorogenic Dye and the wavelength of the Reacted Fluorogenic Dye;
(E) using said means for measurement of said fluorescent signals to measure the fluorescent signals at Time Zero in said Aliquot-Dye, said Aliquot-Nutrient-Dye, said optional Aliquot-Inhibitor-Dye and in said optional Aliquot-Blank, at the wavelength of the Fluorogenic Dye and at the wavelength of the Reacted Fluorogenic Dye to yield fluorescent signals at Time Zero;
(F) calculating the Useful RATIO at Time Zero, wherein the Useful RATIO at Time Zero can be selected from the group consisting of RATIO at Time Zero of the Total Microbiological, Optionally Accounting for Interactions with chemicals and Optionally Accounting for Background Interferences Fluorescent Signal of the Reacted Fluorogenic Dye to the Total Microbiological, Optionally Accounting for Interactions with chemicals and Optionally Accounting for Background Interferences, Fluorescent Signal of the Fluorogenic Dye; the RATIO at Time Zero of the Active Microbiological Fluorescent Signal of the Reacted Fluorogenic Dye to the Active Microbiological Fluorescent Signal of the Fluorogemc Dye; and the RATIO at Time Zero of the Inactive Microbiological Fluorescent Signal of the Reacted Fluorescent Dye to the Inactive Microbiological Fluorescent Signal of the Fluorogenic Dye;
(G) waiting for a time period, designated Time Future, and measuring the fluorescent signals in said Aliquot-Dye, said Aliquot-Inhibitor-Dye, said optional Aliquot-Nutrient-Dye and said optional Aliquot-Blank at the wavelength of the Fluorogenic Dye and the Reacted Fluorogenic Dye at Time Future;
(H) calculating the Useful RATIO at Time Future, wherein the Useful RATIO at Time Future is selected from the group consisting of RATIO at Time Future of the Total Microbiological, Optionally Accounting for Interactions with chemicals and Optionally Accounting for Background Interferences Fluorescent Signal of the Reacted Fluorogenic Dye to the Total Microbiological, Optionally Accounting for Interactions with chemicals and Optionally Accounting for Background Interferences, Fluorescent Signal of the Fluorogenic Dye; the RATIO at Time Future of the Active Microbiological Fluorescent Signal of the Reacted Fluorogenic Dye to the Active Microbiological Fluorescent Signal of the Fluorogenic Dye and the RATIO at Time Future of the Inactive Microbiological Fluorescent Signal of the Reacted Fluorescent Dye to the Inactive Microbiological Fluorescent Signal of the Fluorogenic Dye;
(I) comparing the Useful RATIO at Time Future to the Useful RATIO at Time Zero; and
(J) using the comparison of the Useful RATIO at Time Future to the Useful RATIO at Time Zero to monitor the extent of microbiological contamination in said opaque medium.
The tenth aspect of the instant claimed invention is the process of the ninth aspect of the instant claimed invention further comprising:
(K) using said comparison of the Useful RATIO at Time Future to the Useful RATIO at Time Zero to determine the optimal amount of biocide to deliver to said opaque medium; and
(L) delivering said optimal amount of biocide to the opaque medium.